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DNA mutations of various types often affect the cellular localization and function of gene products. The role of mutant transcripts in the pathogenesis of human disease is increasingly recognized. Among the pathogenic RNA variants are transcripts with single nucleotide substitutions, small insertions or deletions, aberrantly or alternatively spliced transcripts and RNAs derived from fused genes. To discriminate among transcripts, particularly those of low abundance, showing small or large sequence differences, a highly sensitive and specific RNA imaging method is required. The method that fulfills these criteria is single-molecule fluorescence in situ hybridization (smFISH) combined with probes discriminating among RNA variants. With this method, RNA transcripts produced from individual alleles can be imaged, and differences in their transcription, processing, cellular localization and decay can be revealed. In addition to its applications for studying physiological processes involving RNA variants, smFISH offers several advantages for disease related mutation research. Further development of allele-specific microscopic methods may broaden group of RNA variants analyzed, including RNAs with expanded repeat tract, different variants of 3′UTR, RNAs differing in length of polyA tract or transcripts produced from alternative start codons. Moreover, first attempts for allele-specific RNA live imaging were made adding time-lapse analysis. In this review, we discuss important aspects of the variant-specific smFISH methodology and present examples of its applications in deciphering RNA-mediated pathogenic mechanisms in a variety of human diseases, including cancer, neurological, immunological and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Abstract: I formulated a systematic approach to screen for colicin resistance-associated genes by using an attenuated colicin mutant and demonstrated its utility in a screen of genes related to colicin E5 resistance. Screening of an Escherichia coli genome library revealed rstA as a partial resistance gene to colicin E5. Transcript expression of BtuB and OmpF, proteins responsible for translocation of nuclease E colicins, was clearly inhibited in an rstA-overexpressing strain. In addition, the tatA::recN fusion gene provides resistance to an attenuated colicin E5 mutant. Improving tatA::recN by directed coevolution, I created a novel gene with enhanced resistance to colicin E5 and concluded that attenuated colicin-based screening is useful for the discovery and creation of novel colicin resistance-associated genes. [Copyright &y& Elsevier]

A significant percentage of enterotoxigenic Escherichia coli (ETEC) diarrheal disease worldwide is caused by colonization factor antigen I-producing isolates of ETEC either as infant diarrhea with high mortality or traveler’s diarrhea. Bacterial adhesion to intestinal epithelial cells and elaboration of enterotoxins are the main steps in pathogenesis of ETEC, and antibodies to these antigens have been associated with protection. Colonization factor antigen I (CFA/I) is the most frequently encountered adhesin in epidemiological studies. In this study, a DNA fragment encoding the first 25 amino acids of the N-terminal end of CFA/I B subunit was genetically fused to the C-terminal end of the B subunit of heat-labile enterotoxin gene of Escherichia coli with five glycine residues as a linker and expressed in E. coli. The N-terminal fragment was selected because it is relatively conserved among several ETEC fimbriae and strongly reacts with monoclonal antibodies against CFA/I, and several B-cell epitopes have been mapped to this region. The B subunit of heat labile enterotoxin (LTB) of E. coli is a potent immunogen and also elicits antitoxin response. The recombinant protein was recognized by anti-LTB MAb and anti-rCfaB polyclonal antibody and bound GM1 gangliosides receptors in enzyme-linked immunosorbant assay (ELISA). Polyclonal antibody raised in rabbit reacted with the dissociated as well as intact fimbriae expressed on the surface of bacteria in western blot and ELISA, respectively, and blocked fluid accumulation in the rabbit ileal loop assay. This demonstrates that the rLTB-CfaB(1–25) protein could be used for production of anti-LTB and anti-CfaB and that combining the two fragments has not impaired the function of either part. Moreover, complete lack of fluid accumulation in rabbit ileal loops might have resulted from both anti-adhesive and anti-toxin activity of the antibodies raised against the fusion protein. [ABSTRACT FROM AUTHOR]

Abstract: The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity. [Copyright &y& Elsevier]

The human repetin gene is a member of the“fused” gene family and localized in the epidermal differentiation complex on chromosome 1q21. The“fused” gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope (CE). Here, we report the isolation and characterization of the human repetin gene and of its protein product. The repetin protein of 784 amino acids contains EF (a structure resembling the E helix-calcium-binding loop-F helix domain of parvalbumin) hands of the S100 type and internal tandem repeats typical for CE precursor proteins, a combination which is characteristic for“fused” proteins. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium, the inner hair root sheat and in the filiform papilli of the tongue. Ultrastructurally, repetin is a component of cytoplasmic non-membrane“keratohyalin” F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin. Finally, we show that EF hands are functional and reversibly bind Ca2+. Our results indicate that repetin is indeed a member of the fused gene family similar to the prototypical members profilaggrin and trichohyalin. [ABSTRACT FROM AUTHOR]

The protein encoded by theC1orf10gene was described to be esophageal-specific and a marker for cancer development. This protein, however, has the previously unreported structural features of the“fused gene” family combining sequences and structural similarities of both the S100 proteins and precursor proteins of the cornified cell envelope as in profilaggrin, trichohyalin, and repetin. Since all members of this family are expressed in keratinocytes, we suspected a role in epidermal differentiation and named the protein cornulin. Here, we report that human cornulin mRNA is expressed primarily in the upper layers of differentiated squamous tissues including the epidermis. Using polyclonal peptide antibodies, we show that cornulin is expressed in the granular and lower cornified cell layers of scalp epidermis and foreskin, as well as in calcium-induced differentiated cultured keratinocytes. Ca2+-overlay assay indicated that EF-hand domains of cornulin are functional and bind calcium. In HeLa cells, cornulin, co-transfected with transglutaminase 1, was diffusely distributed throughout the cytoplasm in contrast to small proline-rich 4, which localized to the cell periphery. We conclude that cornulin is a new member of the“fused gene” family, does not appear to be a precursor of the cornified cell envelope by itself, and is a marker of late epidermal differentiation. [ABSTRACT FROM AUTHOR]

This study was undertaken to examine the effects of hydrocortisone injected into male mice on the phenotypic expression and inheritance of the Fused ( Fu) gene in their offspring. Data were obtained indicating that there is a hydrocortisone-susceptible period during spermatogenesis. Hydrocortisone injections of males during this period resulted in a statistically significant decrease in the proportion of phenotypically Fu offspring. Genetic analysis with the use of the closely linked recessive marker tufted ( tf) demonstrated that the deficit of phenotypically Fu individuals among offspring is not caused by the differential death of gametes, zygotes or embryos. According to genetic data, this deficit is due to a decrease in the penetrance of the Fu gene and partly to its inherited inactivation. The possible mechanisms of the observed phenomenon are discussed. [ABSTRACT FROM AUTHOR]

Gene fusion is caused by the linkage of previously separate genes or sequences. Recently, an increasing number of novel fusion genes have been identified and associated with tumor progression, and several of them have been suggested as promising targets for tumor therapy. However, there are hardly any studies reporting the association of fusion genes with the progression of oral squamous cell carcinoma (OSCC). In this study, we identified a total of 11 fused genes in OSCC cells. We further analyzed the structure of one fused gene, TRIM52-RACK1, and detected its function in tumor progression in vitro. We found that TRIM52-RACK1 was caused by a deletion of 181,257,187-181,247,386 at 5q35.3 and it promoted OSCC cell proliferation, migration, and invasion. Therefore, TRIM52-RACK1 can be a promising target for tumor therapy in OSCC. • Eleven fused genes were found in OSCC cells. • Fused gene, TRIM-RACK1, was caused by the deletion of 181,257,187-181,247,386 at 5q35.3. • TRIM52-RACK1 promotes OSCC cells proliferation, migration and invasion. [ABSTRACT FROM AUTHOR]

Objective: To analyze the copy number variation of CYP21A2 gene in 21-hydroxylase deficiency (21-OHD) patients, and identify the three copy repetition, single copy deletion of CYP21A2 gene and the type and proportion of CYP21A1P / CYP21A2 fused gene in 21-OHD patients. Methods: A total of 424 patients (140 males and 284 females) with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and the average age was (17.1±12.4) years. All clinical and biochemical data were collected. DNAs were extracted from peripheral blood leukocytes, and CYP21A2 gene mutation and copy number variation were detected by Sanger sequencing and multiple ligation probe amplification (MLPA). Results: Of 424 21-OHD patients, 287 (67.7%) had two copies of CYP21A2 gene, 137 (32.3%) had copy number variation, of which 1 patients (0.2%) had 3 copies of CYP21A2 gene and 136 (32.1%) were carriers of large deletion/rearrangement mutation of CYP21A2 gene. Three pathogenic mutations including a truncated Q319X protein mutation were detected in the patient with 3 copies of CYP21A2 gene. Of 136 patients with large deletion/rearrangement mutation of CYP21A2 gene, 82 (60.3%) carried fused CYP21A1P / CYP21A2 gene, and the remaining 54 harbored the one allele deletion of CYP21A2 . The most common types of fused CYP21A1P / CYP21A2 gene were CH-5, CH-1 and CH-2, with the frequency being 31.7% (26 cases), 26.8% (22 cases) and 19.5% (16 cases), respectively, and followed by CH-4 and CH-7, with the incidence being 8.5% (7 cases) and 4.9% (4 cases), respectively. In addition, two cases of CH-3, CH-6 and CH-8 and one case of CH-9 were detected. Conclusions: This is the first study to detect the occurrence of CYP21A2 gene copy number variation and fused CYP21A1P / CYP21A2 gene in a large cohort of 21-OHD patients. The number of CYP21A2 gene copies in 21-OHD patients includes 2 copies, 1 copy deletion and 3 copies duplication. One copy deletion of CYP21A2 includes one allele deletion of CYP21A2 gene and fused CYP21A1P / CYP21A2 gene. In patients with 3 copies of CYP21A2 gene, pathogenic mutations should be verified in all 3 copies of CYP21A2 gene to make the precise diagnosis. Therefore, the accurate molecular diagnosis of 21-OHD patients should take both genotype and copy number variation of CYP21A2 into account.

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET., (Copyright © 2015 Elsevier Inc. All rights reserved.)